PROJECT SUMMARY Nanoscale vesicles form the structural framework of organelles such as lysosomes, endosomes, exosomes, endocytic and exocytic vesicles, as well as the lipid envelope for viruses. These physiological or pathological nanocarriers are nature?s delivery systems for molecules and therefore represent prototypes for developing novel drug/gene delivery systems for pharmaceutical applications. An important aspect of vesicles is that their mechanical properties allow them to achieve seemingly diametrical tasks: 1- to deform and merge with target membranes to deliver their cargos, and 2- to maintain physical integrity without rupturing in dynamic biological environments. Pure synthetic vesicles (i.e. liposomes), for example, do not possess sufficient mechanical integrity to effectively withstand harsh perturbations present in biological environments (e.g. large fluctuations in static pressure), but reinforcement by more complex structural features, such as membrane proteins and protein-lipid complexes, can provide structural integrity, while maintaining the ability to deform and to fuse with target membranes. Hence, studying the mechanical properties of vesicles, and understanding the mechanisms of reinforcement, as well as the effect of soluble effectors on vesicles? mechanics at the nanoscale, is of great significance for both fundamental and applied purposes. Not only can it help us understand the fundamental biological transport phenomena, but also can lead to new solutions for bio-inspired drug/gene delivery systems. Force spectroscopy of liposomes is the most direct way to understand mechanical properties of vesicles, however, with the current technologies it is very challenging to do force spectroscopy on nanoscale liposomes in solution. The current state-of-the-art technique, atomic force spectroscopy (AFM) is expensive and time- consuming, is low-throughput, and requires highly-trained operators and complex sample preparation. Furthermore, there is currently no method that can separate and sort vesicles based on their mechanical properties, limiting our ability to directly compare mechanical properties with functional characteristics. In this project we will develop a nanopore based force spectroscopy method, that overcomes limitations of AFM, to characterize the mechanical properties of nanoscale liposomes and can sort liposomes based on their mechanical properties. Two specific aims of this are Aim 1: to detect and measure varied mechanical properties of nanoscale vesicles using resistive pulse sensing in solid-state nanopores, and Aim 2: to develop an automated feedback-controlled system that can sort nanovesicles based on their mechanical properties. The proposed nanopore force spectroscopy can be used to characterize mechanical properties of naturally- occurring nanovesicals such as viruses, exosomes, etc. In addition, an automated feedback-controlled system will be developed that can separate samples of desired mechanical properties (e.g. rigidity) out of a mixed population of vesicles with varied properties. Such a platform can be used to sort heterogeneous biological samples based on their mechanical behavior and study the functional role of biomechanics at the nanoscale.